An efficient method to uniquely identify every individual would have value in quality control and sample tracking of large collections of cell lines or DNA as is now often the case with whole genome ...association studies. Such a method would also be useful in forensics. SNPs represent the best markers for such purposes. We have developed a globally applicable resource of 92 SNPs for individual identification (IISNPs) with extremely low probabilities of any two unrelated individuals from anywhere in the world having identical genotypes. The SNPs were identified by screening over 500 likely/candidate SNPs on samples of 44 populations representing the major regions of the world. All 92 IISNPs have an average heterozygosity >0.4 and the F st values are all <0.06 on our 44 populations making these a universally applicable panel irrespective of ethnicity or ancestry. No significant linkage disequilibrium (LD) occurs for all unique pairings of 86 of the 92 IISNPs (median LD = 0.011) in all of the 44 populations. The remaining 6 IISNPs show strong LD in most of the 44 populations for a small subset (7) of the unique pairings in which they occur due to close linkage. 45 of the 86 SNPs are spread across the 22 human autosomes and show very loose or no genetic linkage with each other. These 45 IISNPs constitute an excellent panel for individual identification including paternity testing with associated probabilities of individual genotypes less than 10⁻¹⁵, smaller than achieved with the current panels of forensic markers. This panel also improves on an interim panel of 40 IISNPs previously identified using 40 population samples. The unlinked status of the subset of 45 SNPs we have identified also makes them useful for situations involving close biological relationships. Comparisons with random sets of SNPs illustrate the greater discriminating power, efficiency, and more universal applicability of this IISNP panel to populations around the world. The full set of 86 IISNPs that do not show LD can be used to provide even smaller genotype match probabilities in the range of 10⁻³¹-10⁻³⁵ based on the 44 population samples studied.
We have completed a second-generation linkage map that incorporates sequence-based positional information. This new map, the Rutgers Map v.2, includes 28,121 polymorphic markers with physical ...positions corroborated by recombination-based data. Sex-averaged and sex-specific linkage map distances, along with confidence intervals, have been estimated for all map intervals. In addition, a regression-based smoothed map is provided that facilitates interpolation of positions of unmapped markers on this map. With nearly twice as many markers as our first-generation map, the Rutgers Map continues to be a unique and comprehensive resource for obtaining genetic map information for large sets of polymorphic markers.
We have developed a next-generation sequencing assay to quantify biomarkers of the host immune response in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. This assay aims to provide ...clinicians with a comprehensive characterization of the immunologic tumor microenvironment as a guide for therapeutic decisions on patients with solid tumors. The assay relies on RNA-sequencing (seq) to semiquantitatively measure the levels of 43 transcripts related to anticancer immune responses and 11 transcripts that reflect the relative abundance of tumor-infiltrating lymphocytes, as well as on DNA-seq to estimate mutational burden. The assay has a clinically relevant 5-day turnaround time and can be conducted on as little as 2.5 ng of RNA and 1.8 ng of genomic DNA extracted from three to five standard FFPE sections. The standardized next-generation sequencing workflow produced sequencing reads adequate for clinical testing of matched RNA and DNA from several samples in a single run. Assay performance for gene-specific sensitivity, linearity, dynamic range, and detection threshold was estimated across a wide range of actual and artificial FFPE samples selected or generated to address preanalytical variability linked to specimen features (eg, tumor-infiltrating lymphocyte abundance, percentage of necrosis), and analytical variability linked to assay features (eg, batch size, run, day, operator). Analytical precision studies demonstrated that the assay is highly reproducible and accurate compared with established orthogonal approaches.
Objective: In isolated populations, ‘background’ linkage disequilibrium (LD) has been shown to extend over large genetic distances. This and their reduced environmental and genetic heterogeneity has ...stimulated interest in their potential for association mapping. We compared LD unit map distances with pair-wise measurements of LD in a dense single nucleotide polymorphism (SNP) set. Methods: We genotyped 771 SNPs in an 8 Mb segment of chromosome 22 on 101 individuals from the isolated village of Talana, Sardinia, and compared with outbred European populations. Results: Heterozygosity was remarkably similar in both populations. In contrast, the extent of LD observed was quite different. The decay of LD with distance is slower in the isolate. The differences in LD map lengths suggest that useful LD extends up to three times farther in the Sardinian population; smaller differences are seen with pairwise LD metrics. While LD map length slightly decreases with average relatedness, cryptic relatedness does not explain the decrease in LD map length. Haplotypes, block boundaries, and patterns of LD are similar in both populations, suggesting a shared distribution of recombination hotspots. Conclusions: About 15% fewer haplotype tagging SNPs need to be genotyped in the isolate, and possibly 70% fewer if selecting SNPs evenly spaced on the metric LD map.
DNA microarrays are rapidly becoming a fundamental tool in discovery-based genomic and biomedical research. However, the reliability of the microarray results is being challenged due to the existence ...of different technologies and non-standard methods of data analysis and interpretation. In the absence of a "gold standard"/"reference method" for the gene expression measurements, studies evaluating and comparing the performance of various microarray platforms have often yielded subjective and conflicting conclusions. To address this issue we have conducted a large scale TaqMan Gene Expression Assay based real-time PCR experiment and used this data set as the reference to evaluate the performance of two representative commercial microarray platforms.
In this study, we analyzed the gene expression profiles of three human tissues: brain, lung, liver and one universal human reference sample (UHR) using two representative commercial long-oligonucleotide microarray platforms: (1) Applied Biosystems Human Genome Survey Microarrays (based on single-color detection); (2) Agilent Whole Human Genome Oligo Microarrays (based on two-color detection). 1,375 genes represented by both microarray platforms and spanning a wide dynamic range in gene expression levels, were selected for TaqMan Gene Expression Assay based real-time PCR validation. For each platform, four technical replicates were performed on the same total RNA samples according to each manufacturer's standard protocols. For Agilent arrays, comparative hybridization was performed using incorporation of Cy5 for brain/lung/liver RNA and Cy3 for UHR RNA (common reference). Using the TaqMan Gene Expression Assay based real-time PCR data set as the reference set, the performance of the two microarray platforms was evaluated focusing on the following criteria: (1) Sensitivity and accuracy in detection of expression; (2) Fold change correlation with real-time PCR data in pair-wise tissues as well as in gene expression profiles determined across all tissues; (3) Sensitivity and accuracy in detection of differential expression.
Our study provides one of the largest "reference" data set of gene expression measurements using TaqMan Gene Expression Assay based real-time PCR technology. This data set allowed us to use an alternative gene expression technology to evaluate the performance of different microarray platforms. We conclude that microarrays are indeed invaluable discovery tools with acceptable reliability for genome-wide gene expression screening, though validation of putative changes in gene expression remains advisable. Our study also characterizes the limitations of microarrays; understanding these limitations will enable researchers to more effectively evaluate microarray results in a more cautious and appropriate manner.
We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw ...accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.
Patients with severe coronavirus disease 2019 (COVID-19) develop a febrile pro-inflammatory cytokinemia with accelerated progression to acute respiratory distress syndrome (ARDS). Here we report the ...results of a phase 2, multicenter, randomized, double-blind, placebo-controlled trial of intravenous (IV) plasma-purified alpha-1 antitrypsin (AAT) for moderate to severe ARDS secondary to COVID-19 (EudraCT 2020-001391-15).
Patients (n = 36) were randomized to receive weekly placebo, weekly AAT (Prolastin, Grifols, S.A.; 120 mg/kg), or AAT once followed by weekly placebo. The primary endpoint was the change in plasma interleukin (IL)-6 concentration at 1 week. In addition to assessing safety and tolerability, changes in plasma levels of IL-1β, IL-8, IL-10, and soluble tumor necrosis factor receptor 1 (sTNFR1) and clinical outcomes were assessed as secondary endpoints.
Treatment with IV AAT resulted in decreased inflammation and was safe and well tolerated. The study met its primary endpoint, with decreased circulating IL-6 concentrations at 1 week in the treatment group. This was in contrast to the placebo group, where IL-6 was increased. Similarly, plasma sTNFR1 was substantially decreased in the treatment group while remaining unchanged in patients receiving placebo. IV AAT did not definitively reduce levels of IL-1β, IL-8, and IL-10. No difference in mortality or ventilator-free days was observed between groups, although a trend toward decreased time on ventilator was observed in AAT-treated patients.
In patients with COVID-19 and moderate to severe ARDS, treatment with IV AAT was safe, feasible, and biochemically efficacious. The data support progression to a phase 3 trial and prompt further investigation of AAT as an anti-inflammatory therapeutic.
ECSA-2020-009; Elaine Galwey Research Bursary.
Purpose
To assess whether preoperative radiologically defined lean muscle measures are associated with adverse clinical outcomes in patients undergoing elective surgery for colorectal cancer.
Methods
...This retrospective UK-based multicentre data collection study identified patients having had colorectal cancer resection with curative intent between January 2013 to December 2016. Preoperative computed-tomography (CT) scans were used to measure psoas muscle characteristics. Clinical records provided postoperative morbidity and mortality data.
Results
This study included 1122 patients. The cohort was separated into a combined group (patients with both sarcopenia and myosteatosis) and others group (either sarcopenia or myosteatosis, or neither). For the combined group, anastomotic leak was predicted on univariate (OR 4.1, 95% CI 1.43–11.79;
p
= 0.009) and multivariate analysis (OR 4.37, 95% CI 1.41–13.53;
p
= 0.01). Also for the combined group, mortality (up to 5 years postoperatively) was predicted on univariate (HR 2.41, 95% CI 1.64–3.52;
p
< 0.001) and multivariate analysis (HR 1.93, 95% CI 1.28–2.89;
p
= 0.002). A strong correlation exists between freehand-drawn region of interest-derived psoas density measurement and using the ellipse tool (
R
2
= 81%;
p
< 0.001).
Conclusion
Measures of lean muscle quality and quantity, which predict important clinical outcomes, can be quickly and easily taken from routine preoperative imaging in patients being considered for colorectal cancer surgery. As poor muscle mass and quality are again shown to predict poorer clinical outcomes, these should be proactively targeted within prehabilitation, perioperative and rehabilitation phases to minimise negative impact of these pathological states.
Preclinical models A1 Intratumoral immunotherapy. B16-F10 murine melanoma model J. Ženka1, V. Caisová1, O. Uher1, P. Nedbalová1, K. Kvardová1, K. Masáková1, G. Krejčová1, L. Paďouková1, I. ...Jochmanová2, K. I. Wolf3, J. Chmelař1, J. Kopecký1 1Department of Medical Biology, Faculty of Science, University of South Bohemia, Česke Budějovice, Czech Republic; 21st Department of Internal Medicine, Medical Faculty of P. J. Šafárik University in Košice, Košice, Slovakia; 3University of Michigan Medical Center, Ann Arbor, MI, United States Correspondence: J. Ženka Background: Cancer immunotherapy based on direct intratumoral injection of immunomodulators has been established at the end of 19th century using Coley’s toxin. Our novel therapeutic strategy is based on the intratumoral injection of optimized mixture of TLR agonists, causing strong inflammatory infiltration. Infiltrating cells (mainly phagocytes) are directed to artificially opsonized tumor cells covered by phagocytosis stimulating ligands. Materials and methods: Immunotherapy was tested using B16-F10 murine melanoma model. Inflammatory infiltration was achieved using the mixture of resiquimod, poly(I:C), and lipoteichoic acid. Artificial opsonisation of tumor cells was elicited by mannan anchored to cell membranes using a hydrophobic anchor. The course of tumor infiltration was studied using flow cytometry. Cytotoxic effect of infiltrating immune cells on opsonized tumor cells was studied in vitro. Participation of acquired immunity was elucidated on the basis of intracellular IFN-gamma production by lymphocytes. Results: Optimized cancer immunotherapy based on the synergy of TLR agonists with artificially induced tumor opsonisation resulted in complete cure of 83% of mice with advanced melanomas. Moreover, cured mice acquired resistance to retransplantation of tumor cells. Applied therapy has a strong antimetastatic effect. In the first phase of immunotherapy, granulocyte predominance was observed, followed by involvement of acquired immunity. Conclusions: Intratumoral immunotherapy initiated by innate immunity based attack followed by joining of mechanisms of acquired immunity is a promising approach for future application in clinical practice as compounds used are safe for humans. A2 Assessing humanized mouse models for cancer immunotherapy L. Loumagne, J. Mestadier, S. D’agostino, A. Rohaut, Y. Ruffin, V. Croize, O. Lemaître, S. S. Sidhu Sanofi, Vitry, France Correspondence: L. Loumagne Background: Preclinical models that can recapitulate a functional human immune system are essential tools for the continued investigation of novel immunotherapy approaches. In this regard, immunodeficient mice offer a unique opportunity to reconstitute, at least partially, a human immune system together with human tumors to characterize immunomodulator compounds in a more physiologically relevant setting. The aim of this project was to perform a head to head comparison of several highly immunodeficient mouse strains engrafted with human CD34+ Hematopoietic Stem Cells (HSCs) for their ability to develop and sustain a multi-lineage engraftment of human immune cells. The most promising mouse model(s) will then be assessed for their capacity to support human tumor xenografts (CDX and PDX). Finally, we will determine if we can elicit an immune response to selected immunotherapeutic agents to drive anti-tumor activity. Materials and methods: The mice strains analyzed are NSG, NOG, NSG-SGM3, and NOG-EXL; the latter two being transgenic for the expression of human cytokines important in myeloid cell development. Humanization was performed in-house by injecting human umbilical cord blood CD34+ HSCs from different donors into the tail-vein of 4–5 week old female mice post-irradiation. To monitor human immune system reconstitution, flow cytometry analysis on blood was performed at approximately 4 week intervals beginning at week 4 post-injection of human CD34+ HSCs. At week 18, a terminal analysis was performed, consisting of a thorough immune cell profiling of blood, spleen and bone marrow by flow cytometry. Tissues were collected for the immunohistochemical staining of human immune cells and ex vivo functional assays will be performed. Results: Comparison of the immune system from different humanized mouse models show that all strains display high levels of engraftment of human CD45+ cells ranging from 50 to 70% at 18 weeks post-humanization. Population such as T CD4+ and CD8+ as well as B cells are well reconstituted and minor populations such as monocytes/macrophages, NK, and subsets of DC are also present at a lower percentage. Complete results obtained from different mouse strains will be available on the congress day. Conclusions: Overall, this project will provide a head-to-head comparison of several humanized mouse models that will be of value in the immunotherapy field according to the potent advantages of these models and the paucity of studies directly comparing different immunodeficient mouse strains. A3 A predictive PD-L1*CD8 cell density score for NSCLC patients treated with durvalumab S. Althammer1, K. Steele2, M. Rebelatto2, T. Tan1, T. Wiestler1, A. Spitzmueller1, R. Korn1, G. Schmidt1, B. Higgs2, X. Li2, L. Shi2, X. Jin2, K. Ranade2 1DEFINIENS, Muenchen, Germany; 2Medimmune, Gaithersburg, MD, United States Correspondence: S. Althammer Background: Current PD-1/PD-L1 checkpoint inhibitor based immuno-therapies for advanced non-small cell lung cancer (NSCLC) show impressive response rates and long survival. We used automated image1 and data analysis of immunohistochemically (IHC) stained tissue sections within the Tissue Phenomics methodology to determine whether CD8+ and PD-L1+ cells densities could better identify patients most likely to respond to durvalumab than using a manual PD-L1 scoring of positive tumor cells (25% cutoff). Durvalumab is a human IgG1 monoclonal antibody that inhibits programmed death ligand-1 (PD-L1) binding to programmed death-1 and B7.1/CD80, restoring antitumor immunity2,3. Materials and methods: CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating durvalumab in advanced NSCLC and other solid tumors4. Digital slides from 163 baseline tumor biopsies, IHC stained for PD-L1 (Ventana SP263) and CD8 (Ventana SP239), were fully automatically scored with Definiens’ Developer XD 2.1.4 software using the product of PD-L1+ and CD8+ cell densities. The image analysis results were quality controlled using ground truth annotations provided by a panel of three pathologists. A discovery set (n = 84) was used to identify the scoring algorithm and the cutoff value, which was then tested on a validation set (n = 79). The robustness of the scoring algorithm was pre-validated using Monte-Carlo (MC) cross-validation. Results: The most accurate and robust scoring method in terms of MC performance metrics (positive predictive value, prevalence, pOS, pPFS) was identified as the product of average CD8+ and PD-L1+ cells densities in the pathologist annotated tumor center. Patients with high CD8*PD-L1 scores show significantly improved outcome compared to the CD8*PD-L1 low subgroup (ORR: 0.39 vs 0.07, OS log rank p-value: 0.0099 (training set) and 0.00053 (confirmation set)). CD8*PD-L1 further appears to predict patients’ outcome better than the PD-L1 status visually determined by pathologists (ORR: 39% vs 27%, median OS: 24.3 vs 17.8). Conclusions: Although further validation studies are required, we observe that a scoring algorithm based on automated image analysis of CD8+ and PDL1+ cell densities in baseline tumor biopsies improves the stratification of NSCLC patients treated with durvalumab when compared to visual histopathology scoring. References 1. Brieu, et al. Proc. SPIE 9784 Medical Imaging 2016: doi:10.1117/12.2208620. 2. MedImmune/AstraZeneca. Data on file. 3. Ibrahim R, et al. Semin Oncol 2015;42(3):474–83. 4. Rizvi NA, et al. J Clin Oncol 2015;33(Suppl.):Abstract 8032. A4 The influence of specific cytokines on cancer microtissue infiltrating cytotoxic T lymphocyte subpopulations S. Koeck1, A. Amann1, G. Gamerith1, M. Zwierzina2, E. Lorenz3, H. Zwierzina1, J. Kern3 1Medical University of Innsbruck, Department of Internal Medicine V, Innsbruck, Austria; 2Medical University of Innsbruck, Department of Plastic, Reconstructive and Aesthetic Surgery, Innsbruck, Austria; 3Tyrolean Cancer Research Institute, Innsbruck, Austria Correspondence: S. Koeck Background: Cancer associated fibroblasts are known to highly interact with infiltrating immune cells within the tumor microenvironment. In this work, we investigate the influence of specific fibroblast derived cytokines on the infiltration capacity of CD3 + CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture System. Materials and methods: 3D tumor microtissues were cultivated using a hanging drops system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 days to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added for 24 h. Endogenous cytokine expression of interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-12p70, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in microtissue mono-, co- and tri-cultures was analyzed with a multi-cytokine immunoassay. Subsequently, measured cytokines were added to cancer cell/PBMC co-cultures to investigate the effect of these cytokines on infiltrating immune cell subpopulations. Infiltrating subpopulations were investigated by flow cytometry. Results: In both A549/SV80 and Calu-6/SV80 co-cultures and in SV80 monocultures, the cytokines TNF-α, IL-2, IL-5, IL-6 and IL-12p70 were measured. Minimal or no concentrations of the investigated cytokines were detected in cancer cell and PBMC monocultures. IL-4 and IFN-γ were not measured in all approaches. Additional application of the detected cytokines showed that IL-2 inhibited infiltra
PDF
